The long-term objectives of this proposal are to better understand the DNA damage response mechanisms that influence the response of normal cells and cancer cells to chemotherapy. Thymineless stress (TLS) is induced by inhibition of the enzyme, thymidylate synthase (TS), and is the therapeutic effect of several classes of antineoplastic drugs, such as 5-fluorouracil and raltitrexed (Tomudex). Thymineless death is the terminal event resulting from depletion of TTP pools, and is characterized by cessation of replication and DNA fragmentation. Despite decades of study, it is unclear which DNA damage response pathways are activated in response to TLS. Two characteristics are consistently observed in cells that have lost TS function by genetic or pharmacological means. Nucleotide pools are altered, such that an elevation in dUTP and dATP, and a decrease in dGTP and/or dCTP are observed. Secondly, the loss of TS function induces DNA strand breaks, possibly at sites of replication. Damage response mechanisms, such as base excision repair and mismatch repair, proceed via strand break intermediates; thus, activation of these repair pathways may contribute to strand breaks associated with TTP depletion. Moreover, strand breaks are expected to activate recombinational mechanisms, which could induce further genetic instability. The hypotheses to be tested in this project are that DNA repair pathways are involved in the response to TLS and that these pathways potentially influence responses to chemotherapy treatment that induces TLS. The Specific Aims of this project are: Aim 1. To examine the role of base excision repair (BER) and mismatch repair (MMR) in TLS; to identify in vitro biomarkers of TLS that correlate with cytotoxicity of TS inhibitors. Aim 2. To determine whether recombinational mechanisms are activated during TLS. Aim 3. To examine the role of MMR in TLS in mouse models of colon cancer; to determine whether in vitro biomarkers of TLS may be utilized as in vivo biomarkers for chemotherapy-induced thymineless death. Understanding the cellular responses to chemotherapy (in vivo, ultimately) should provide insights into treatments that can exploit differences in damage response mechanisms between normal and neoplastic cells.